Most trace elements function through proteins of which they are constituents. Selenium is incorporated as the amino acid selenocysteine during translation of primary protein structure. Selenocysteine is synthesized and inserted into proteins co-translationally by a complex process.
Regardless of the form in which the selenium is ingested, it is transformed by a variety of metabolic pathways via the same intermediary pool into the specific selenocysteine-containing selenoproteins which are responsible for the biological effects of selenium. The levels of selenocysteine-containing selenoproteins in tissues appear to be controlled by homeostatic means. The common sources of selenium in animal nutrition including selenomethionine, Se-methyl-selenocysteine, selenite, and selenate take different pathways to the intermediary selenium pool which is ultimately incorporated in the specific seleno-proteins or further converted into polar metabolites that can be readily excreted. An overview of selenoprotein biosynthesis is illustrated as FIG. 1.
The steps of seleno protein biosynthesis include (i) Dietary and tissue forms of selenium are converted to selenide; (ii) The enzyme selenophosphate synthetase acts upon the selenide or its closely related form (substrate) to form monoselenophosphate as a product. (iii) Monoselenophosphate is utilized to transform ser-tRNA[ser]Sec to selenocysteine. The selenocysteyl-tRNA[ser]Sec is inserted into the growing polypeptide chain of the selenoprotein by a selenosome complex.
Families of selenoproteins include the glutathione peroxidases, the iodothyronine deiodinases and the thioredoxin reductases. These are redox enzymes that take advantage of the chemical properties of selenium to catalyze, respectively, removal of hydroperoxides by glutathione, deiodination of thyroid hormones and support of cellular processes requiring reduction of disulfides. Several additional selenoproteins have been identified.
An insight into animal selenoproteins in relation to their biological functions is illustrated in the following table.
SelenoproteinKnown FunctionGlutathione peroxidasesHydroperoxide Catabolism [Arthur, J. R. (2000) The glutathioneperoxidases. Cell. Mol. Life Sci. 57: 1825-1835.]Sperm structure [Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri,A., Wissing, J. & Flohe', L. (1999) Dual function of the selenoproteinPHGPx during sperm maturation. Science 285: 1393-1396.][Pfeifer H., Conrad M., Roethein D., Kyriakopoulos A., Brielmeier M.,Bornkamm G. W., Behne D. Identification of a Specific Sperm NucleiSelenoenzyme Necessary for Protamine Thiol Cross-Linking During SpermMaturation. FASEB J 15: 1236-1238 (2001)]Thioredoxin reductasesProtein thiol redox regulation [Arne' r, E. S. J. & Holmgren, A. (2000)Physiological functions of thioredoxin and thioredoxin reductase. Eur. J.Biochem. 267: 6102-6109.]Vitamin C recycling [May, J. M., Mendiratta, S., Hill, K. E. & Burk, R. F.(1997) Reduction of dehydroascorbate to ascorbate by the selenoenzymethioredoxin reductase. J. Biol. Chem. 272: 22607-22610.]Synthesis of DNA [Arne' r, E. S. J. & Holmgren, A. (2000) Physiologicalfunctions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267:6102-6109.]IodothyronineT4 activation, T3 inactivation [Bianco, A. C., Salvatore, D., Gereben, B.,deiodinasesBerry, M. J. & Larsen, P. R. (2002) Biochemistry, cellular and molecularbiology, and physiological roles of the iodothyronine selenodeiodinases.Endocr. Rev. 23: 38-89.Methionine sulfoxideRemoval of reactive oxygen species through methionineReductase B[Moskovitz, J., Singh, V. K., Requena, J., Wilkinson, B. J., Jayaswal, R. K.& Stadtman, E. R. (2002) Purification and characterization of methioninesulfoxide reductases from mouse and Staphylococcus aureus and theirsubstrate stereo specificity. Biochem. Biophys. Res. Commun. 290: 62-65.Selenoprotein WAntioxidant [Sun, Y., Gu, Q. P. & Whanger, P. D. (2001) SelenoproteinW in over expressed and underexpressed rat glial cells in culture. J. Inorg.Biochem. 84: 151-156.]Selenoprotein PSelenium transport [Burk, R. F., Hill, K. E., Read, R. & Bellew, T. (1991)Response of rat selenoprotein P to selenium administration and fate of itsselenium. Am. J. Physiol. 261: E26-E30.]Hill, K. E., Zhou, J. D., McMahan, W. J., Motley, A. K., Atkins, J.,Gesteland, R. & Burk, R. F. (2002) Characterization of selenoprotein Pknockout mice. FASEB J. 16: A605.Antioxidant [Burk, R. F., Hill, K. E., Awad, J. A., Morrow, J. D., Kato, T.,Cockell, K. A. & Lyons, P. R. (1995) Pathogenesis of diquat-induced livernecrosis in selenium deficient rats. Assessment of the roles of lipidperoxidation by measurement of F2 isoprostanes. Hepatology 21: 561-569.]
The importance of dietary selenium in the maintenance of human and animal health has been well established in the prior art.
The recognition of the essential role of selenium in human and animal nutrition has resulted in the establishment of a Recommended Daily Allowance (RDA) for humans and approval of the inclusion of additional selenium compounds in animal feed. Recently, the Food and Nutrition Board of the Institute of Medicine revised the RDA for selenium to 55 [mu] g [Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. Washington, D.C.: National Academy Press, (2000)]. These inorganic selenium salts can be added at the level of 0.3 ppm Se in feed dry matter. In June 2000, the FDA approved the use of selenium yeast in poultry broiler and layer diets.
Several studies have demonstrated that selenium is more bioavailable from organic sources than from inorganic sources. Foremost among them are L-selenomethionine and L-Se-methylselenocysteine.
These synthetic seleno-amino acids pose certain challenges for cosmetic and topical uses such (a) low water solubility; (b) crystals bearing water repellant properties; (c) low rate of dissolution; and (d) sometimes malodorous properties. To overcome these shortcomings, the present invention describes several short dipeptides incorporating separately L-selenomethionine and L-Se-methyl-selenocyteine, especially with L-glutamic acid, in particular the γ-L-glutamyl analogs.
The synthesis of γ-L-Glutamyl-Se-methyl-L-selenocysteine has been discussed by E. Block, M. Birringer, W. Jiang, T. Nakahoda, H. J. Thompson, P. J. Toscano, H. Uzar, X. Zhang, Z. Zhu in the J Agric. Food Chem., 49, 458 (2001). The method involves coupling the reactants triethylammonium-N-(trityl)-L-γ-glutamate and methyl ester hydrochloride of Se-methyl L-selenocysteine with dicyclohexylcarbodiimide. The resulting material is denuded of the protecting ester and triphenyl groups through the successive use of acid and alkali followed by an ion exchange column chromatographic purification. This method bears innate disadvantages like (a) being circuitous and involving complex steps like column purifications (b) not being adaptable to a large scale industrial manufacture (c) Unfavorable economics of a production method.
It is thus the principle object of the present invention to develop a novel synthetic method for the production of selenoproteins, wherein the said method.
Will confer useful properties to the selenopeptide product that will help overcome inherent disadvantages associated with synthetic selenoacids; is simple to perform and adaptable to larger scale industrial manufacture; will consume less time to perform; andWill ensure a pure selenopeptide product that can be readily used without any need for further purification.
It is another object of the present invention to develop isomeric peptides of L-selenomethionine and Se-Methyl-L-selenocysteine through a novel synthetic process that would result in:
Stable, solid isomeric peptides of L-selenomethionine and Se-Methyl-L-selenocysteine enhanced water solubility of the isomeric peptides of L-selenomethionine and Se-Methyl-L-selenocysteine; enhanced rate of dissolution of the isomeric peptides of L-selenomethionine and Se-Methyl-L-selenocysteine in water; enhanced bioavailability of the isomeric peptides of L-selenomethionine and Se-Methyl-L-selenocysteine; and enhanced purity of the isomeric peptides of L-selenomethionine; Se-Methyl-L-selenocysteine which makes the peptides ready for use without any need for further purification; and isomeric peptides of L-selenomethionine and Se-Methyl-L-selenocysteine which are not malodorous and are suitable cosmeceutical use.
It is another object of the present invention to evaluate isomeric peptides of L-selenomethionine and Se-Methyl-L-selenocysteine developed by a novel synthetic method for useful cosmeceutical properties such as vascular endothelial growth factor promoting activity and anti-5-alpha reductase activity.
It is another object of the present invention to develop cosmeceutical compositions comprising the isomeric peptides of L-selenomethionine and Se-Methyl-L-selenocysteine obtained by a novel synthetic method and which, are marked by (a) purity; (b) enhanced water solubility; (c) enhanced bioavailability; (d) excellent vascular endothelial growth factor promoting activity (e) excellent anti-5-alpha-reductase activity; and (f) the ability to stimulate “hair growth” and prevent/reduce “hair fall” thereby maintaining a perfect homeostasis for hair care.
It is another object of the present invention to develop pharmaceutical compositions comprising the isomeric peptides of L-selenomethionine and Se-Methyl-L-selenocysteine obtained by a novel synthetic method and which, are marked by (a) purity; (b) enhanced water solubility; (c) enhanced bioavailability; (d) excellent vascular endothelial growth factor promoting activity; (e) excellent anti-5-alpha-reductase activity; and (f) the ability to stimulate “hair growth” and prevent/reduce “hair fall” thereby maintaining a perfect homeostasis for hair care.
The present invention fulfills all these objectives and provides further related advantages.